Protocol for obtaining Vigna unguiculata (L.) Walp. transformants carrying editing constructs

Background. The method of agrobacterium mediated transformation is most often used for the transformation of higher plant cells. The choice of explant type, the composition of nutrient media for the selection and regeneration of transformants are important. Cowpea Vigna unguiculata (L.) Walp. is a legume crop. It is recalcitrant for transformation due to its low regeneration after agrobacterial inoculation. The search for genotypes with a high regeneration ability, as well as the creation of an effective protocol for optimal Agrobacterium tumefaciens-mediated transfection protocol are urgent tasks for the delivery of editing system components. The aim of this study is to develop an effective protocol for obtaining cowpea transformants carrying editing constructs. Material and methods. The development of the efficient protocol for obtaining cowpea transformants for the delivery of editing system components was carried out using accessions from the VIR collection. Cotyledonary node parts were used as explants formed by longitudinal incision of the cotyledon node in order to increase the wound surface area. The agrobacterium mediated transformation was performed using a vector on the base of pKSE401 with components of the CRISPR/Cas9 editing system. Organogenesis was induced on MS nutrient medium with phytohormones. The article describes a step-by-step protocol for the efficient production of fertile cowpea transformants. Results and discussion. We experimentally confirmed the organogenetic capacity of the cowpea cotyledonary node to produce shoots in vitro, as well as the possibility of using them as explants for agrobacterium mediated transformation. The frequency of fertile transformants was 6.2% for k-642 genotype. A comparison of the transformation efficiency with the data from previous studies on the cowpea agrobacterium mediated transformation indicates a better yield of transformants based on our proposed protocol. Since the protocol has been validated in the experiment with a vector carrying components of the CRISPR/Cas9 editing system, it can be recommended for use in studies on the production of edited cowpea plants. Conclusion. The obtained results of the agrobacterium mediated transformation of cowpea modified type explants indicate the possibility of successful use of the presented protocol for the genetic transformation of this crop. The k-642 genotype was efficient not only at the stages of regeneration and transformation, but also at the stages of rooting and subsequent plant adaptation to non-sterile conditions. This genotype can be recommended for further fundamental cowpea studies using reverse genetics methods.

1. Behura R., Kumar S., Saha B., Panda M.K., Dey M., Sadhukhan A., Mishra S., Alam S., Sahoo D.P., Sugla T., Sahoo L. Cowpea [Vigna unguiculata (L.) Walp.]. In: Methods in Molecular Biology. Vol. 1223. K. Wang (ed.) Agrobacterium Protocols. Vol. 1, Ch. 20. New York, NY: Springer Humana Press; 2014. p.255-264. DOI: 10.1007/978-1-4939-1695-5_20

2. Bett B., Gollasch S., Moore A., Harding R., Higgins T.J.V. An improved transformation system for cowpea (Vigna unguiculata L. Walp) via sonication and a kanamycin-geneticin selection regime. Frontiers in Plant Science. 2019;10:219. DOI: 10.3389/fpls.2019.00219

3. Burlyaeva M.O., Gurkina M.V., Chebukin P.A Screening of long-podded cowpea (Vigna unguiculata subsp. sesquipedalis (L.) Verdc.) samples from VIR collection for resistance to biotic and abiotic stressors (Skrining obraztsov sparzhevoy vigny (Vigna unguiculata subsp. sesquipedalis (L.) Verdc.) iz kollektsii VIR na ustoychivost' k abioticheskim i bioticheskim stressoram). Breeding and seed production of vegetable crops = Selektsiya i semenovodstvo ovoshchnykh kul’tur. 2014;45:131-141. [In Russian]

4. Che P., Chang S., Simon M.K., Zhang Z., Shaharyar A., Ourada J., O’Neill D., Zhang Z., Torres-Mendoza M., Guo Y., Marasigan K.M, Vielle-Calzada J.-P., Ozias-Akins P., Albertsen M.C., Jones T.J. Developing a rapid and highly efficient cowpea regeneration, transformation and genome editing system using embryonic axis explants. The Plant Journal. 2021;106(3):817-830. DOI: 10.1111/tpj.15202

5. Efremova O.S., Shkryl Y.N., Veremeichik G.N. Regeneration potential in vitro of soybean varieties in agrobacterial transformation. Agrarian bulletin of Primorye. 2017;4(8):21-23. [in Russian]

6. Gerasimova S.V., Kolosovskaya E.V., Vikhorev A.V., Korotkova A.M., Hertig C.W., Genaev M.A., Domrachev D.V., Morozov S.V., Chernyak E.I., Shmakov N.A., Vasiliev G.V., Kochetov A.V., Kumlehn J., Khlestkina E.K. WAX INDUCER 1 Regulates β-Diketone Biosynthesis by Mediating Expression of the Cer-cqu gene cluster in barley. International Journal of Molecular Sciences. 2023;24:6762. DOI: 10.3390/ijms24076762

7. Ignacimuthu S. Agrobacterium mediated transformation of Vigna sesquipedalis Koern (asparagus bean). Indian journal of experimental biology. 2000;38(5):493–498.

8. Integrated DNA Technologies. PrimerQuest™ Tool. Available from: https://eu.idtdna.com/pages/tools/primerquest [accessed Febr. 01, 2024].

9. Ji J., Zhang C., Sun Z., Wang L., Duanmu D., Fan Q. Genome editing in cowpea Vigna unguiculata using CRISPR-Cas9. International Journal of Molecular Sciences. 2019;20(10):2471. DOI: 10.3390/ijms20102471

10. Juranić M., Nagahatenna D.S.K., Salinas-Gamboa R., Hand M.L., Sánchez León N., Leong W.H., How T., Bazanova N., Spriggs A., Vielle Calzada J.-P., Koltunow A.M.G. A Detached leaf assay for testing transient gene expression and gene editing in cowpea (Vigna unguiculata [L.] Walp.). Plant Methods. 2020;16:88. DOI: 10.1186/s13007-020-00630-4

11. Jyothishwaran G., Kotresha D., Selvaraj T., Srideshikan S.H., Rajvanshi P.K., Jayabaskaran C. A modified freeze-thaw method for efficient transformation of Agrobacterium tumefaciens. Current Science. 2007;93(6):770-772.

12. Krylova E.A., Burlyaeva M.O., Tvorogova V.E., Khlestkina E.K. Contrast relative humidity response of diverse cowpea (Vigna unguiculata (L.) Walp.) genotypes: deep study using RNAseq approach. International Journal of Molecular Sciences. 2024;25:11056. DOI: 10.3390/ijms252011056

13. Mao J.Q., Zaidi M.A., Arnason J.T., Altosaar I. In vitro regeneration of Vigna unguiculata (L.) Walp. cv. Blackeye cowpea via shoot organogenesis. Plant Cell Tissue Organ Culture. 2006;87:121-125. DOI: 10.1007/s11240-006-9145-8

14. Murashige T., Skoog F.A. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum. 1962;15:473-497. DOI: 10.1111/j.1399-3054.1962.tb08052.x

15. Muthukumar B., Mariamma M., Veluthambi K., Gnanam A. Genetic transformation of cotyledon explants of cowpea (Vigna unguiculata L. Walp) using Agrobacterium tumefaciens. Plant Cell Report. 1996;15:980-985. DOI: 10.1007/BF00231600

16. Pal J.K., Kumar S., Singh M. Genetic transformation of Vigna unguiculata tissue by Agrobacterium tumifaciens. Indian Journal of Genetics and Plant Breeding. 2011;71(1):84-86.

17. Popelka J.C., Gollasch S., Moore A., Molvig L., Higgins T.J. Genetic transformation of cowpea (Vigna unguiculata L.) and stable transmission of the transgenes to progeny. Plant Cell Reports. 2006;25(4):304-312. DOI: 10.1007/s00299-005-0053-x

18. Pratap A., Prajapati U., Singh C.M., Gupta S., Rathore M., Malviya N., Tomar R., Gupta A.J., Tripathi S., Singh N.P. Potential, constraints and applications of in vitro methods in improving grain legumes. Plant Breeding. 2018;137:235-249. DOI: 10.1111/pbr.12590

19. Razgonova M.P., Burlyaeva M.O., Zinchenko Y.N., Krylova E.A., Chunikhina O.A., Ivanova N.M., Zakharenko A.M., Golokhvast K.S. Identification and spatial distribution of bioactive compounds in seeds Vigna unguiculata (L.) Walp. by laser microscopy and tandem mass spectrometry. Plants. 2022;11:2147. DOI: 10.3390/ plants11162147

20. Sahoo L., Sugla T., Jaiwal P.K. In vitro regeneration and genetic transformation of cowpea, mungbean, urdbean and azuki bean In: P.K. Jaiwal; R.P. Singh (eds). Applied Genetics of Leguminosae Biotechnology. Dordrecht; Boston: Kluwer Academic Publishers; 2003. p.89-120.

21. Somers D.A., Samac D.A., Olhoft P.M. Recent advances in legume transformation. Plant Physiology. 2003;131:892-899. DOI: 10.1104/pp.102.017681

22. Ukhatova Y.V., Erastenkova M.V., Korshikova E.S., Krylova E.A., Mikhailova A.S., Semilet T.V., Tikhonova N.G., Shvachko N.A., Khlestkina E.K. Improvement of crops using the CRISPR/Cas System: new target genes. Molecular Biology. 2023;57(3):375-397. DOI: 10.1134/S0026893323030135

23. Xing H.-L., Dong L., Wang Z.-P., Zhang H.-Y., Han C.-Y., Liu B., Wang X.-C., Chen Q.-J. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biology. 2014;14:327. DOI: 10.1186/s12870-014-0327-y